Summary High-level drug resistance puts TB patients at increased risk of treatment failure. However, patients also fail treatment with apparently drug susceptible strains even in clinical trial settings where compliance is well monitored. We postulate that decades of drug exposure have selected for bacterial strains that have alterations in drug-mediated clearance that are not identified by conventional drug resistance testing and that these changes put compromise patient outcomes. In previous work, we identified mutations that alter bacterial drug susceptibility in ways that are not identified by conventional clinical resistance testing but are revealed by assaying bacterial drug responses using time-dependent killing assays under host-like environmental conditions. We hypothesize that these mutations predispose to treatment failure even as they also accelerate the emergence of high-level drug resistance. To test this hypothesis, we propose to test the hypothesis that mutations associated with resistance and treatment failure in clinical cohorts alter Mtb survival in the presence of antibiotics. For this aim, we will construct and phenotype a barcoded library of isogenic strains carrying mutations identified in our analyses of clinical strains as associated with drug resistance or treatment failure. We will test the hypothesis that the drug resistance-associated mutations and mutations associated with treatment failure cause treatment failure in a mouse model of infection. Finally, because many resistance associated mutations appear to cause multidrug tolerance, we will assess their effects on the efficacy of new antibiotics and small molecules in late stage development for Mtb.